TNFα receptor-derived binding protein

ABSTRACT

A polypeptide is provided which is capable of binding human TNFα and which has the first three cysteine-rich subdomains, but not the fourth cysteine-rich subdomain, of the extracellular binding domain of a receptor selected from the group consisting of the 55 kD and 75 kD receptors for human TNFα. The ability of the polypeptide to bind TNFα means that it can be used for treating diseases mediated by TNFα activity, such as rheumatoid arthritis.

The present invention relates to recombinant proteins and their use.

Tumour necrosis factor-α (TNFα) is a potent cytokine which elicits abroad spectrum of biological responses. TNFα causes the cytolysis orcytostasis of many tumour cell lines in vitro, induces the haemorrhagicnecrosis of transplanted tumours in mice, enhances the phagocytosis andcytotoxicity of polymorphonuclear neutrophils, and modulates theexpression of many proteins, including lipoprotein lipase, class Iantigens of the major histo-compatibility complex, and cytokines such asinterleukin 1 and interleukin 6. TNFα appears to be necessary for anormal immune response, but large quantities produce dramatic pathogeniceffects. TNFα has been termed "cachectin" since it is the predominantfactor responsible for the wasting syndrome (cachexia) associated withneoplastic disease and parasitemia. TNF is also a major contributor totoxicity in gram-negative sepsis, since antibodies against TNF canprotect infected animals.

The many activities of TNFα are mediated by binding to a cell surfacereceptor. Radioligand binding studies have confirmed the presence of TNFreceptors on a wide variety of cell types. Although these receptors areexpressed in limited numbers (1,000-10,000 receptors/cell), they bindTNFα with high affinity (Ka=10⁹ M⁻¹ at 4° C.). Lymphotoxin (LT, alsotermed TNFβ) has similar, if not identical, biological activities toTNFα, presumably because both are recognized by the same receptor.

Recently, several laboratories have detected heterogeneity in TNFreceptor preparations. Two distinct cell surface receptors which bindTNFα and TNFβ have recently been characterised at the molecular level.cDNA for one form of the receptor with a Mr of 55 kD was isolatedutilising probes designed from the peptide sequence of a soluble form ofthe receptor (1,2). A second receptor of Mr 75 kD was cloned by a COScell expression approach (3). Both receptors are members of a largerfamily of cytokine receptors which include the nerve growth factorreceptor, the B cell antigen CD40, the rat T cell antigen MRC OX40. Inaddition these receptors are homologous to the predicted product of atranscriptionally active open reading frame from shope fibroma viruswhich appears to give rise to a secreted protein.

The most conserved feature amongst this group of cell surface receptorsis the cysteine rich extracellular ligand binding domain, which can bedivided into four repeating motifs of about forty amino acids. We havenow generated four soluble receptor derivatives of the 55 kD TNFαreceptor (TNFR). Each derivative is composed of the extracellularbinding domain but without one of the cysteine rich subdomains. We havefound that the derivative which lacks the membrane-proximal fourthsubdomain retains the ability to bind TNFα with high affinity. Thisfinding has general applicability.

Accordingly, the present invention provides a polypeptide which iscapable of binding human TNFα and which consists essentially of:

(a) the first three cysteine-rich subdomains, but not the fourthcysteine-rich subdomain, of the extracellular binding domain of the 55kD or 75 kD receptor for human TNFα; or

(b) an amino acid sequence having a homology of 90% or more with thesaid sequence (a).

The invention also provides:

a DNA sequence which encodes such a polypeptide;

a vector which incorporates a DNA sequence of the invention and which iscapable, when provided in a transformed host, of expressing thepolypeptide of the invention encoded by the DNA sequence; and

a host transformed with such a vector.

In the accompanying drawings:

FIG. 1 shows the nucleotide sequence of the human TNFα cDNA (SEQ ID NO:24) and encoded amino acid (SEQ ID NO: 25) sequence. The predictedsignal sequence residues are numbered -40 to -1. The transmembranedomain is boxed and potential N-linked glycosylation sites areoverlined. The sequence homologous with the designed oligonucleotideprobe is found at nucleotide positions 477-533.

FIG. 2 is a Northern blot (lanes 1-3) of 10 μg of oligo-dT selected RNAfrom human 293 cells (fibroblast cell line) (lane 1), placenta (lane 2)and spleen (lane 3) hybridised with the TNF receptor cDNA (Smal-EcoRIfragment). The Southern blot (lanes 4-6) was hybridized with the sameprobe. Human genomic DNA (5 μg per lane) was digested with Pst1 (lane4), Hind III (lane 5) and EcoRI (lane 6).

FIG. 3 shows the binding characteristics of recombinant human TNFreceptor expressed in COS-7 cells. The direct binding of recombinant ¹²⁵I-TNFα to COS-7 cells transfected with prTNFR is presented in panel A.The inset contains Scatchard analysis derived from this data. As shownin panel B, monolayers of Cos-7 cells transfected with TNFR cDNA wereincubated with 1 nM ¹²⁵ I-TNF in the presence of various concentrationsof unlabelled TNFα or TNFβ.

FIG. 4 shows the effects of soluble TNFR on TNFα binding and biologicalactivity. Panel A shows the effects of supernatants from Cos-7 cellstransfected with a cDNA encoding a soluble form of the TNF receptor(pTNFRecd, closed circles) or mock transfected (open circles) on ¹²⁵I-TNF binding to U937 cells. Panel B shows the effects of thesesupernatants on TNF mediated killing of WEHI 164 (clone 13) line. Assayswere performed as described in Materials and Methods.

FIG. 5 is a diagram of the DNA sequence of pTNFRecd and is also astrategy map for polymerase chain reaction (PCR)-based domain deletion,in which 5'UTR is the 5'-untranslated region and I to IV are the fourcysteine-rich subdomains. The oligonucleotides employed in PCR in theExample and relevant restriction sites are also shown.

FIG. 6 shows lined up the amino acid sequences of the four cysteine-richsubdomains of the 55 kD (TNFR-55) and 75 kD (TNFR-75) receptors and ofrat nerve growth factor receptor (NGFR), human CD40 and rat OX40.Homology is shown by means of boxes.

FIGS. 7 to 11 show the nucleotide sequence and the predicted amino acidsequence of the encoded polypeptide of pTNFRecd, pΔI, pΔII, pΔIII andpΔIV.

FIG. 12 shows the results of the assays described in the Example 1.

FIG. 13 shows diagrammatically the DNA encoding the 75 kD receptor inwhich I to IV are the four cysteine-rich subdomains. Oligonucleotidesemployed in PCR-domain deletion are also shown.

A polypeptide according to the invention is capable of binding humanTNFα. Typically the polypeptide has a binding affinity for human TNFα of10⁷ M⁻¹ or greater, for example 10⁸ M⁻¹ or greater. The affinity may befrom 10⁷ to 10¹⁰ M⁻¹, for example from 10⁸ to 10⁹ M⁻¹.

A preferred polypeptide consists essentially of the first threecysteine-rich subdomains of the extracellular binding domain of the 55kD receptor for human TNFα. The sequence (a₁) of these three subdomainsis: V C P Q G K Y I H P Q N N S I C C T K C H K G T Y L Y N D C P G P GQ D T D C R E C E S G S F T A S E N H L R H C L S C S K C R K E M G Q VE I S S C T V D R D T V C G C R K N Q Y R H Y W S E N L F Q C F N C S LC L N G T V H L S C Q E K Q N T V C (SEQ ID NO: 4).

A useful polypeptide has the amino acid sequence (c): M G L S T V P D LL L P L V L L E L L V G I Y P S G V I G L V P H L G D R E K R D S V C PQ G K Y I H P Q N N S I C C T K C H K G T Y L Y N D C P G P G Q D T D CR E C E S G S F T A S E N H L R H C L S C S K C R K E M G Q V E I S S CT V D R D T V C G C R K N Q Y R H Y W S E N L F Q C F N C S L C L N G TV H L S C Q E K Q N T V C T (SEQ ID NO: 2).

In an alternative embodiment, the polypeptide may consist essentially ofthe first three cysteine-rich subdomains of the extracellular bindingdomain of the 75 kD receptor.

Apart from the amino acid sequence (a), the polypeptides mayalternatively consist essentially of an amino acid sequence (b) having ahomology of 90% or more with sequence (a). The degree of homology may be95% or more or 98% or more. Amino acid sequence (a) may therefore bemodified by one or more amino acid substitutions, insertions and/ordeletions and/or by an extension at either or each end. There should beno modification of the cysteine-residues, however. A polypeptidecomprising sequence (b) must of course still be capable of binding humanTNFα.

For example, one or more amino acid residues of the sequence (a), otherthan a cysteine residue, may be substituted or deleted or one or moreadditional amino acid residues may be inserted; provided thephysicochemical character of the original sequence is preserved, i.e. interms of charge density, hydrophobicity/hydrophilicity, size andconfiguration. Conservative substitutions may be made. Candidatesubstitutions are, based on the one-letter code (Eur. J. Biochem. 138,9-37, 1984):

A for G and vice versa,

V by A, L or G;

K by R;

S by T and vice versa;

E for D and vice versa; and

Q by N and vice versa.

Up to 15 residues may be deleted from the N-terminal and/or C-terminalof the polypeptide, for example up to 11 residues or up to 5 residues.

The polypeptides of the invention consist essentially of sequence (a) or(b). They do not contain a fourth cysteine-rich subdomain. However, thepolypeptides may be longer polypeptides of which sequence (a) or (b) isa part. A short sequence of up to 50 amino acid residues may be providedat either or each terminal of sequence (a) or (b). The sequence may haveup to 30, for example up to 20 or up to 10, amino acid residues.

Alternatively, a much longer extension may be present at either or eachterminal of sequence (a) or (b) of up to, for example, 100 or 200 aminoacid residues. Longer amino acid sequences may be fused to either oreach end. A chimaeric protein may be provided in which the or eachextension is a heterologous amino acid sequence, i.e. a sequence notnaturally linked to the amino acid sequence above. Such a chimaericprotein may therefore combine the ability to bind specifically to humanTNFα with another functionality.

The polypeptides of the invention lack the fourth cysteine-richsubdomain of the 55 kD or 75 kD receptor as the case may be. Inparticular, they lack the cysteine residues of the fourth subdomain.They therefore do not comprise, immediately after the thirdcysteine-rich subdomain, any of the amino acid sequence up to the lastcysteine residue of the fourth cysteine-rich subdomain of the relevantreceptor except possibly the first amino acid residue of that sequence.The polypeptides may extend beyond that first amino acid residue asindicated above, though, by way of other amino acid sequences.

The polypeptides are typically recombinant polypeptides, although theymay be made by synthetic methods such as solid-phase or solution-phasepolypeptide synthesis in which case an automated peptide synthesiser maybe employed. They may therefore commence with a N-terminal residue M.They are prepared by recombinant DNA technology. The preparation of thepolypeptides therefore depends upon the provision of a DNA sequenceencoding the polypeptide. A suitable sequence encoding the first threecysteine-rich subdomains of the extracellular binding domain of the 55kD receptor comprises: GTG TGT CCC CAA GGA AAA TAT ATC CAC CCT CAA AATAAT TCG ATT TGC TGT ACC AAG TGC CAC AAA GGA ACC TAC TTG TAC AAT GAC TGTCCA GGC CCG GGG CAG GAT ACG GAC TGC AGG GAG TGT GAG AGC GGC TCC TTC ACCGCT TCA GAA AAC CAC CTC AGA CAC TGC CTC AGC TGC TCC AAA TGC CGA AAG GAAATG GGT CAG GTG GAG ATC TCT TCT TGC ACA GTG GAC CGG GAC ACC GTG TGT GGCTGC AGG AAG AAC CAG TAC CGG CAT TAT TGG AGT GAA AAC CTT TTC CAG TGC TTCAAT TGC AGC CTC TGC CTC AAT GGG ACC GTG CAC CTC TCC TGC CAG GAG AAA CAGAAC ACC GTG TGC (SEQ ID NO: 3).

A DNA sequence may further comprise a DNA sequence encoding a signalsequence fused to the 5' end of the coding sequence. Any signal sequencemay be appropriate. The signal sequence should be capable of directingsecretion of the polypeptide of the invention from the cell in which thepolypeptide is expressed. The signal sequence may be the natural signalsequence for the 55 kD TNFα receptor. An appropriate DNA sequenceencoding the first three cysteine-rich subdomains of the extracellularbinding domain of the 55 kD receptor and such a signal sequence istherefore: ATG GGC CTC TCC ACC GTG CCT GAC CTG CTG CTG CCG CTG GTG CTCCTG GAG CTG TTG GTG GGA ATA TAC CCC TCA GGG GTT ATT GGA CTG GTC CCT CACCTA GGG GAC AGG GAG AAG AGA GAT AGT GTG TGT CCC CAA GGA AAA TAT ATC CACCCT CAA AAT AAT TCG ATT TGC TGT ACC AAG TGC CAC AAA GGA ACC TAC TTG TACAAT GAC TGT CCA GGC CCG GGG CAG GAT ACG GAC TGC AGG GAG TGT GAG AGC GGCTCC TTC ACC GCT TCA GAA AAC CAC CTC AGA CAC TGC CTC AGC TGC TCC AAA TGCCGA AAG GAA ATG GGT CAG GTG GAG ATC TCT TCT TGC ACA GTG GAC CGG GAC ACCGTG TGT GGC TGC AGG AAG AAC CAG TAC CGG CAT TAT TGG AGT GAA AAC CTT TTCCAG TGC TTC AAT TGC AGC CTC TGC CTC AAT GGG ACC GTG CAC CTC TCC TGC CAGGAG AAA CAG AAC ACC GTG TGC ACC (SEQ ID NO: 1).

A DNA sequence encoding a polypeptide of the invention may besynthesised. Alternatively, it may be constructed by isolating a DNAsequence encoding the 55 kD or 75 kD receptor from a gene library anddeleting DNA downstream of the coding sequence for the first threecysteine-rich subdomains of the extracellular binding domain of thereceptor. This gives DNA encoding the first three subdomains of eitherreceptor. As an intermediate step, DNA encoding the entire or nearly theentire extracellular binding domain may be isolated and digested toremove DNA downstream of the coding sequence for the first threesubdomains.

A modified nucleotide sequence, for example encoding an amino acidsequence (b), may be obtained by use of any appropriate technique,including restriction with an endonuclease, insertion of linkers, use ofan exonuclease and/or a polymerase and site-directed mutagenesis.Whether a modified DNA sequence encodes a polypeptide of the inventioncan be readily ascertained. The polypeptide encoded by the sequence canbe expressed in a suitable host and tested for its ability to bindspecifically human TNFα.

For expression of a polypeptide of the invention, an expression vectoris constructed. An expression vector is prepared which comprises a DNAsequence encoding a polypeptide of the invention and which is capable ofexpressing the polypeptide when provided in a suitable host. Appropriatetranscriptional and translational control elements are provided,including a promoter for the DNA sequence, a transcriptional terminationsite, and translational start and stop codons. The DNA sequence isprovided in the correct frame such as to enable expression of thepolypeptide to occur in a host compatible with the vector.

The expression vector is then provided in an appropriate host. Cellsharbouring the vector are grown so as to enable expression to occur. Thevector may be a plasmid or a viral vector. Any appropriate host-vectorsystem may be employed.

The transformed host may be a prokaryotic or eukaryotic host. Abacterial or yeast host may be employed, for example E. coli or S.cerevisiae. Insect cells can alternatively be used, in which case abaculovirus expression system may be appropriate. As a furtheralternative, cells of a mammalian cell line, such as Chinese HamsterOvary (CHO) Cells may be transformed. A polypeptide glycosylated at one,two or three of the sites shown in FIG. 1 can be obtained by suitablechoice of the host cell culture.

The polypeptide of the invention can be isolated and purified. TheN-terminal of the polypeptide may be heterogeneous due to processing ofthe translation product within a cell or as the product is beingsecreted from a cell. A mixture of polypeptides according to theinvention, having different N-terminii, may therefore be obtained. Thepolypeptide is soluble.

The polypeptides of the invention have activity binding human TNFα. Thisactivity is indictive of the possible use of the polypeptides in theregulation of TNFα-mediated responses by binding and sequestering humanTNFα, for example possible use in treatment of pulmonary diseases,septic shock, HIV infection, malaria, viral meningitis, graft versushost reactions and autoimmune diseases such as rheumatoid arthritis.

For this purpose, a polypeptide of the present invention may beformulated in a pharmaceutical composition. The pharmaceuticalcomposition also comprises a pharmaceutically acceptable carrier ordiluent.

The polypeptide of the invention may be administered to a patient by anyconvenient route. The choice of whether an oral route or a parenteralroute, such as subcutaneous, intravenous or intramuscularadministration, is adopted; of the dose; and of the frequency ofadministration depends upon a variety of factors. These factors includethe purpose of the administration, the age and weight of the patientbeing treated and the condition of the patient. Typically, however, thepolypeptide is administered in an amount of from 1 to 1000 μg per dose,more preferably from 10 to 100 μg per dose, for each route ofadministration.

The following Examples illustrate the invention. A Reference Example isprovided.

REFERENCE EXAMPLE

1. Materials and Methods Reagents

Recombinant human TNFα and TNFβ were supplied as highly purifiedproteins derived from E. coli. The specific activities of thesepreparations were approximately 10⁷ units/mg, as measured in the murineL929 cell cytotoxicity assay (4). The synthetic oligonucleotides wereprepared by Oswel DNA Service (University of Edinburgh).

Isolation of TNFα 55 kD receptor cDNA clones

The sequence of a peptide fragment (E M G Q V E I S S T V D R D T V C G)(SEQ ID NO: 5) of the TNF binding protein was used to design a syntheticoligonucleotide probe (5' AAG GAG ATG GGC CAG GTT GAG ATC TCT TCT ACTGTT GAC AAT GAC ACT GTG TGT GGC-3' (SEQ ID NO: 6)). The 57-mer DNA probewas labelled with ³² P and T4 polynucleotide kinase (New England Biolab,Beverly, Mass.) and used to screen a placenta cDNA library in gt10(5,6). Approximately 800,000 phage were transferred to nitrocellulosefilters and screened at reduced stringency (7). Filters were incubatedfor 2 hours at 42° C. in 0.05M sodium phosphate, pH 6.5, 20% formamide,0.75M sodium chloride, 0.075M sodium citrate, 1% polyvinyl pyrrolidone(Sigma, St Louis, Mo.), 1% Ficoll, 1% bovine serum albumin (Sigma), and50 ng/ml sonicated salmon sperm DNA (Sigma). The radiolabelled probe wasthen added to the filters (10⁸ cpm/ml final concentration) which werehybridized for 16 hours. Filters were washed extensively in 0.06M sodiumchloride, 0.006M sodium citrate, 1% SDS at 37° C. and positive cloneswere identified by autoradiography. Ten hybridizing clones were plaquepurified (5) and cDNA insert size was determined by polyacrylamide gelelectrophoresis of EcoRI digested phage DNA. The inserts of two cDNAclones were sequenced using the dideoxy chain termination technique (8).

Southern and Northern blot analysis

DNA was isolated from human lymphocytes by the method of Blin andStafford (9) and used for Southern blot analysis (10). DNA was digestedwith restriction endonucleases (New England Biolabs), fractionated on a1% agarose gel, and transferred to nitrocellulose. Hybridization andwashing were conducted under stringent conditions (6) using a ³²p-labelled preparation of a 600 bp fragment of the TNF receptor cDNA.Northern blot analysis was performed (11) on oligo-dT selected RNAisolated from human placenta, spleen (generously provided by theCooperative Human Tissue Network, Birmingham, Ala.) and a fibroblastcell line (293 cells). Following electrophoresis on a formaldehyde 1.2%agarose gel, the RNA was transferred to nitrocellulose and hybridizedwith the TNFα receptor DNA probe under stringent conditions.

Mammalian cell expression of the human TNFα 55 kD receptor andderivatives

The coding region of the majority of the human TNFα 55 kD receptor wasisolated as an EcoRI fragment and cloned into a mammalian cellexpression vector (12), resulting in plasmid prTNFR. The EcoRI fragmentencodes 374 amino acids of the TNF receptor; the 81 carboxyl terminalresidues of the cytoplasmic domain are therefore missing from thisplasmid construction. A derivative of the TNFα receptor was produced byengineering a termination codon just prior to the transmembrane domain.The polymerase chain reaction (PCR) technique (13) was used to generatea 300 bp restriction fragment containing a BgIII site at the 5' end anda HindIII site preceded by a TAG stop codon at the 3' end. The PCRprimers were 5'GCTGCTCCAAATGCCGAAAG (SEQ ID NO: 7)and5'AGTTCAAGCTTTTACAGTGCCCTTAACATTCTAA (SEQ ID NO: 8).

The PCR product was gel purified and cloned into the TNF receptorexpression plasmid (described above) digested with BgIII and HindIII.DNA sequencing confirmed that the resulting plasmid (pTNFRecd) containedthe designed DNA sequence. E. coli harbouring pTNFRecd were deposited atthe National Collection of Industrial and Marine Bacteria, Aberdeen, GBon 11 Sep. 1990 under accession number NCIMB 40315.

The TNFα receptor expression plasmids were transfected into monkey COS-7cells using Lipofectin (Gibco BRL, Bethesda, Md.) according to themanufacturer's instructions. Cells were cultured in Dulbecco's modifiedEagle's medium containing 10% fetal calf serum.

Analysis of recombinant TNFα 55 kD receptor derivatives

TNFα was radioiodinated with the Iodogen method (Pierce) according tothe manufacturer's directions. The specific activity of the ¹²⁵ I-TNFαwas 10-30 μCu/μg. COS cells transfected with the TNFα receptor cDNA(prTNFR, 1300 bp EcoRI fragment) were incubated for 24 hours and thenseeded into six well tissue culture plates (Nunc) at 4.5×10⁸ cells perwell. The cells were incubated for a further 48 hours and then receptorexpression was quantitated by radioligand binding for 2 hours at 4° C.Non-specific binding of ¹²⁵ I-TNFα was determined in the presence of a1,000 fold molar excess of unlabelled TNFα. Binding data was analysed bythe method of Scatchard (14).

The TNFα receptor derivative was analysed for inhibition of ¹²⁵ I-TNFαbinding to the natural receptor on human U937 cells. Culture supernatantwas harvested 72 hours after COS cells were transfected with pTNFRecd.U937 cells (2×10⁸ cells in 200 μl) were incubated with 1nM ¹²⁵ I-TNFαand dilutions of COS cell media for 2 hours at 4° C. Cells were thencentrifuged through 20% sucrose to remove unbound TNFα. Non-specificbinding was determined in the presence of 1 μM unlabelled TNFα.

The TNFα receptor derivative was also analyzed for inhibition of TNFαcytotoxic effects in vitro. The cytotoxicity assay was performed asdescribed on the TNF sensitive cell line WEHI 164 clone 13 (15). Serialdilutions of supernatants from COS cells transfected with pTNFRecd ormock transfected controls were incubated with a constant amount of TNFα(1 ng/ml) for 1 hour at 27° C. before addition to the assay.

2. RESULTS

Isolation and characterization of the TNFα 55 kD receptor cDNA

A partial amino acid sequence of the TNF binding protein was used todesign a synthetic oligonucleotide probe. The radiolabelled probe wasused to screen a human placenta cDNA library in lambdagt10 and tenhybridizing phage were isolated. The nucleotide and deduced amino acidsequences of the longest cDNA clone are depicted in FIG. 1. The thirdpotential ATG initiation codon occurs at position 156 of the nucleotidesequence; the first two ATG codons are closely followed by terminationcodons, and the third ATG is preceded by the best translation initiationconsensus nucleotides (16). The cDNA encodes an open reading frame of1365 bases which codes for a polypeptide of 455 residues. Both of thepeptide sequences determined by amino acid sequencing were identified inthe encoded cDNA (17 of 19 and 18 of 19 matching residues). The aminoterminal end identified for the TNF binding protein corresponds to thecDNA encoded sequence beginning at residue 41. The first 35 amino acidsare generally quite hydrophobic and probably represent a signalsequence. Residues 35-40 are highly charged (DREKR SEQ ID NO: 7)) andsuch a sequence is not typically found in secretory signal sequences(17); perhaps the natural receptor is processed by proteolysis afterresidue 40 which contains a dibasio cleavage site (KR). Hydropathyanalysis of the protein sequence predicts a single transmembrane domainof 23 amino acids. This hydrophobic sequence divides the protein into anextracellular domain of 171 residues and a cytoplasmic domain of 221residues. The amino acid composition determined for the TNF bindingprotein corresponds well with the predicted composition of theextracellular domain encoded by the cDNA (results not shown). Thediscrepancy between the predicted receptor size (40,000 daltons) and thesize determined by SDS-polyacrylamide gel electrophoresis (65,000daltons, 18-20) is probably due to glycosylation; there are fourpotential N-linked glycosylation sites in the sequence, three of whichare in the extracellular domain. The sequence contains a large number(17) of cysteine residues, 24 of which are in the extracellular domain.The arrangement of these cysteine residues is similar to that of severalother cell surface proteins, suggesting that the TNF receptor isstructurally related to a family of receptors.

A Northern blot analysis is presented in FIG. 2. The ³² P-labelled cDNAhybridized to a single predominant band of oligo-dT selected RNA fromhuman placenta or spleen. A minor larger transcript was also observed inRNA from a fibroblast cell line. The size of the hybridizing species is2400 bases, in good agreement with the size of isolated cDNA. Also shownin FIG. 2 is a Southern blot of human genomic DNA hybridized with a 600bp probe from the cDNA. In each of the three different restrictiondigests, only a single hybridized signal was observed. Thus the TNFreceptor that we have isolated appears to be encoded by a single gene.

Expression of recombinant TNF receptor sequences in mammalian cells

To confirm that the cDNA shown in FIG. 1 indeed encodes the TNFreceptor, the cDNA was engineered for expression in mammalian cells. ThecDNA contains an EcoRI site at position 1270 of FIG. 1. The receptorcoding sequence was isolated as a 1300 bp EcoRI-fragment (containing allbut the last 81 codons of the cytoplasmic domain) and inserted into amammalian cell expression vector containing a cytomegalovirus promoterand SV40 transcription termination sequences (12). The resulting plasmidwas transfected into COS cells which were analyzed for TNF receptorexpression after three days. As shown in FIG. 3, the transfected cellsspecifically bound radioiodinated TNFα in a saturable and dose dependentfashion. The population of COS cells expressed approximately 1×10⁸receptors per cell. The measured binding affinity of recombinantreceptors was 2.5×10⁹ M⁻¹ at 4° C. which is in close agreement withnatural receptor on human cells (19,20). The binding of ¹²⁵ I-TNFα (1nM) to these cells could be inhibited by the addition of unlabelled TNFαor lymphotoxin (FIG. 3b). COS cells transfected with just the expressionvector did not significantly bind ¹²⁵ I-TNFα (less than 2% of thebinding seen with the cDNA transfection).

The extracellular domain of the TNF receptor is naturally shed fromcells. To produce a similar recombinant derivative, a stop codonpreceding the transmembrane domain was engineered into the cDNA by PCRmutagenesis. The modified DNA was inserted into the expression plasmidand subsequently transfected into COS cells. After three days, the COScell media was tested for inhibition of TNFα binding to human U937cells. As shown in FIG. 4a, the transfected cell media inhibited up to70% of the binding of TNFα. The recombinant TNF receptor derivative wasnext tested for inhibition of TNFα biological activity. A sensitivebioassay for TNFα is a measurement of cytolysis of mouse WEHI 164 (clone13) cells. The transfected cell media inhibited 60% of TNFα cytotoxicityon this cell line (FIG. 4b). Media from mock transfected COS cells didnot inhibit TNFα induced cytotoxicity or binding. These experimentsdemonstrate that the recombinant extracellular domain of the TNFreceptor is capable of binding TNF and inhibiting its biologicalactivity.

EXAMPLE 1 Expression of polypeptide consisting essentially of the firstthree cysteine-rich subdomains of the extracellular binding domain ofthe 55 kD receptor

1. MATERIALS AND METHODS

Reagents

E. coli derived recombinant human TNFα had a specific activity of 2×10⁷U/mg in an L929 cytotoxicity assay. Oligonucleotides were purchased fromOswel DNA service (University of Edinburgh).

Generation of the recombinant soluble TNFR derivatives

Deletion of each of the subdomains in the recombinant soluble TNFR wasachieved by means of PCR fragment joining and PCR mutagenesis. Thesequence of the oligonucleotides used in these experiments is given inTable 1 and their locations relative to the four cysteine richsubdomains is shown in FIG. 5. The four subdomains are lined up withrespect to one another in FIG. 6.

The plasmid pTNFRecd (Reference Example) is shown in FIG. 7. pTNFRecdwas further modified to remove 5' untranslated sequences by cloning ofthe Cla I/Bgl II digested product of a PCR using oligos 5' Cla and IIIAinto ClaI/Bgl II digested pTNFRecd, to generate 5'-ΔCla. Digestion of5'-ΔCla with Pst-1 and religation resulted in the generation of pΔII,which lacks the second cysteine rich subdomain (FIG. 9). The fourthcysteine rich subdomain was removed by cloning of the BglII/Hind IIIdigested product of a PCR using oligonucleotides 5A and 4D intoBglII/Hind III 5'-ΔCla; this introduced a termination codon after aminoacid 167 (counting from the initial methionine) to yield pΔIV (FIG. 11).The constructs p I (FIG. 8) and pΔIII (FIG. 10) which lack the first andthird cysteine rich subdomains respectively were generated by joiningPCR fragments by means of overlaps introduced into the primers used forthe PCR. The gel purified products of PCR's using 5' Cla and IA and IBand 5D were mixed and subjected to further amplification using 5' Claand 5D as primers. The resulting fragment was digested with ClaI andBglII and cloned into ClaI/BglII digested pTNFRecd, to yield pΔI.

Similarly the gel purified products of PCR's using 5' Cla and IIIA andIIIB and 5D were mixed and subjected to further amplification using 5'Cla and 5D as primers. This product was digested with BglII and HindIIIand cloned into Bgl II/Hind III cut 5'-ΔCla to yield pΔIII. In all casesthe cloned derivatives were analysed by restriction enzyme analysis andDNA sequencing using sequenase (United States Biochemical Corporation).

                                      TABLE 1                                     __________________________________________________________________________    Structure of the mutagenic oligonucleotides                                   Oligo                                                                         Name                                                                              Sequence                                                                  __________________________________________________________________________    5'Cla                                                                             5'-GTTCTATCGATAAGAGGCCATAGCTGTCTGGC-3'                                                                       (SEQ ID NO: 10)                            IA  5'-GCTCTCACACTCTCTCTTCTCCCTGTCCCCTAG-3'                                                                      (SEQ ID NO: 11)                            IB  5'-AGGGAGAAGAGAGAGTGTGAGAGCGGCTCCTTC-3'                                                                      (SEQ ID NO: 12)                            IIIA                                                                              5'-TGCATGGCAGGTACACACGGTGTCCGGTCCAC-3'                                                                       (SEQ ID NO: 13)                            IIIB                                                                              5'-GACACCGTGTGTACCTGCCATGCAGGTTTCTTT-3'                                                                      (SEQ ID NO: 14)                            4D  5'-GGCCAAGCTTCAGGTGCACACGGTGTTCTG-3'                                                                         (SEQ ID NO: 15)                            5A  5'-GCTGCTCCAAATGCCGAAAG-3'     (SEQ ID NO: 16)                            5D  5'-AGTTCAAGCTTTACAGTGCCCTTAACATTCTAA-3'                                                                      (SEQ ID NO: 17)                            __________________________________________________________________________

Analysis of recombinant soluble TNFR derivatives

COS cells were maintained in Dulbecco's modified Eagles mediumcontaining 5% foetal calf serum. The soluble TNFα receptor derivativeswere transfected into monkey COS cells by means of lipofectin(GIBCO-BRL, Bethesda Md.) according to the manufacturers protocol andcell free supernatants harvested 72 hours post transfection.

Inhibition of TNFα activity

The soluble TNFα receptor derivatives were analyzed for inhibition ofTNFα cytotoxic activity in vitro. The cytotoxicity assay was performedas described on the TNFα sensitive cell line WEHI 164 clone 13. Serialdilutions of supernatants from COS cells transfected with the mutantreceptors or mock transfected controls were incubated with a constantamount of TNF (1 ng/ml) for 1 hour at 37° C. before addition to theassay.

2. RESULTS

In order to understand more about the contribution of the individualcysteine rich subdomains to the binding of TNFα by the soluble form ofthe 55 kD TNF receptor, we removed each subdomain by PCR mutagenesis(FIG. 5). COS cells were transfected with each of these constructs andthe supernatants were assayed for their ability to inhibit the cytotoxicactivity of TNFα. FIG. 12 panel A shows that conditioned medium from COScells tranfected with pTNFRecd inhibits TNFα as previously described.Removal of the fourth cysteine rich subdomain resulted in a proteinwhich, similar to TNFRecd, was a potent inhibitor of TNFα (FIG. 12 panelB). The mutants lacking the first, second and third subdomains did notshow any inhibitory activity in the TNFα cytotoxicity assay.

EXAMPLE 2 Expression of polypeptide consisting essentially of the firstthree cysteine-rich subdomains of the extracellular binding domain ofthe 75 kD receptor

The coding region of the human 75 kD TNFα receptor was isolated from a Tcell lambda ZAP library, using a probe based on published sequences (3)and cloned into the EcoRI site of a mammalian cell expression vector(12) resulting in plasmid p75TNFR. In more detail, RNA was extractedfrom a cell line expressing the 75 kD receptor and reverse transcribed.Any cell line expressing this receptor could be used, such as thosedescribed by Smith et al (3). The product of the reverse transcriptionwas subjected to 25 cycles of PCR using the following primers: 5' CGCAGA ATT CCC CGC AGC CAT GGC GCC CGT CGC C 3' (SEQ ID NO: 18) and 5' GTAAGG ATC CTA TCG CCA GTG CTC CCT TCA GCT 3' (SEQ ID NO: 19).

These primers are directed against the extracellular binding domaincoding region of the 75 kD receptor and were taken from Smith et al (3).The amplified product was gel purified and shown to encode TNFR. Thiswas subsequently used to screen the library. Plaque purification wasperformed essentially as described in the Reference Example except thatthe probe was labelled by random priming (21) and hybridised in 50%formamide. Filters were washed in 0.2×SSC (Standard Saline Citrate)twice at 60° C.

A derivative of the 75 kD TNFα receptor was produced by engineering atermination codon just prior to the transmembrane domain. Referring toFIG. 13, the polymerase chain reaction (PCR) technique was used togenerate a 274 bp restriction fragment containing a BglII site at the 5'end and an Xba I site preceded by a TAG stop codon at the 3' end. ThePCR primers were 5' ACACGACTTCATCCACGGATA (SEQ ID NO: 20) and 5'ACGTTCTAGACTAGTCGCCAGTGCTCCCTTCAGCTG (SEQ ID NO: 21). The PCR productwas digested with Bgl II and Xba I, gel purified and cloned into the TNFreceptor expression plasmid (described above) digested with BglII andXba I. DNA sequencing confirmed that the resulting plasmid contained thedesigned DNA sequence.

A similar approach was utilised to generate a construct which lacked thefourth cysteine-rich subdomain of the 75 kD TNFα receptor. PCR wasperformed using a primer upstream of the Esp I site in the 75 kD TNFRand a primer which introduced a TAG termination codon and an Xba I site.The sequences of the primers was 5' CAG AAC CGC ATC TGC ACC TGC (SEQ IDNO: 22) and 5' ACGTTCTAGACTTGCACACCACGTCTGATGTTTC (SEQ ID NO: 23)respectively. The PCR product was digested with EspI and Xba I and the110 bp DNA fragment gel purified and cloned into Esp I Xba I digestedp75TNFR.

REFERENCES

1. Loetscher, H., Pan, Y.-C. E., Lahm, H.-W., Gentz, R., Brockhaus, M.,Tabuchi, H. and Lesslayer, W. (1990) Cell, 61, 351-359.

2. Schall, T. J., Lewis, M., Koller, K. J., Lee, A., Rice, G. C., Wong,G. H. W., Gatanaga, T., Granger, G. A., Lentz, R., Raab, H., Kohl, W. J.and Goeddel, D. Y. (1990) Cell, 61, 361-370.

3. Smith, C. A., Davis, T., Anderson, D., Solam, L., Beckmann, M. P.,Jerzy, R., Dower, S. K., Cosman, D. and Goodwin, R. G. (1990) Science248, 1019-1023.

4. Ruff, M. R. & Gifford, G. E. (1981) Infection and Immunity, 31, 380.

5. Maniatis, T., Hardison, R. C., Lacy, E., Lauer, J., O'Connell, C.,Quon, D., Sim, G. K. and Efstratiadis, A. (1978) Cell 15, 687-701.

6. Lawn, R. M., Fritsch, E. F., Parker, R. C., Blake, G & Maniatis, T.(1978) Cell 15, 1157-1174.

7. Gray, P. W., Leong, S. R., Fennie, E., Farrar, M. A., Pingel, J. T.and Schreiber, R. D. (1989) Proc. Natl. Acad. Sci USA 86, 8497-8501.

8. Smith, A. J. H., (1980) Meth. Enzym. 65 560-580.

9. Blin, N, & Stanford, D. W. (1976) Nucl. Acids Res. 3, 2303-2398.

10. Southern, E. M. (1975) J. Molec. Biol. 98, 503-517.

11. Dobner, P. R., Kawasaki, E. S., Yu, L. Y. and Bancroft, F. C. (1981)Proc. Natl. Acad. Sci. USA. 78, 2230-2234.

12. Eaton, D. L., Wood, W. I., Eaton, D., Hass, P. E., Hollinghead, P.,Wion, K., Mather, J., Lawn, R. M., Vahar, G. A. and Gorman, C. (1986)Biochemistry 25: 8343-8347.

13. Scharf, S. J., Horn, G. T., Erlich, H. A. (1986) Science 233,1076-1079.

14. Scatchard, G. (1949) Ann. New York Acad. Sci. 51, 660-672.

15. Espevik, T. & Nissen-Meyer, J. (1986) J. Immunol. Meths. 95, 99-105.

16. Kozak, M. (1989) J. Cell. Biol. 108, 229-241.

17. von Heijne, G. (1988) Nucl. Acids. Res. 14, 4683-4690.

18. Creasy, A. A., Yamamoto, R. & Vitt, C. R. (1987) Proc. Natl. Acad.Sci. USA. 84, 3293-3297.

19. Stauber, G. B., Alyer, R. A. & Aggarwal, B. B. (1988) J. Biol. Chem.263, 19098-19104.

20. Scheurich, P., Ucer, U., Kronke, M. and Pfitzenmaier, K. (1986) Int.J. Cancer, 38, 127-133.

21. Feinburg, A. & Vogelstein, B (1984) Analytical Biochem. 137,266-277.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 57                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 501 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..501                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      ATGGGCCTCTCCACCGTGCCTGACCTGCTGCTGCCGCTGGTGCTCCTG48                            MetGlyLeuSerThrValProAspLeuLeuLeuProLeuValLeuLeu                              151015                                                                        GAGCTGTTGGTGGGAATATACCCCTCAGGGGTTATTGGACTGGTCCCT96                            GluLeuLeuValGlyIleTyrProSerGlyValIleGlyLeuValPro                              202530                                                                        CACCTAGGGGACAGGGAGAAGAGAGATAGTGTGTGTCCCCAAGGAAAA144                           HisLeuGlyAspArgGluLysArgAspSerValCysProGlnGlyLys                              354045                                                                        TATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGTGCCACAAA192                           TyrIleHisProGlnAsnAsnSerIleCysCysThrLysCysHisLys                              505560                                                                        GGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGAC240                           GlyThrTyrLeuTyrAsnAspCysProGlyProGlyGlnAspThrAsp                              65707580                                                                      TGCAGGGAGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTC288                           CysArgGluCysGluSerGlySerPheThrAlaSerGluAsnHisLeu                              859095                                                                        AGACACTGCCTCAGCTGCTCCAAATGCCGAAAGGAAATGGGTCAGGTG336                           ArgHisCysLeuSerCysSerLysCysArgLysGluMetGlyGlnVal                              100105110                                                                     GAGATCTCTTCTTGCACAGTGGACCGGGACACCGTGTGTGGCTGCAGG384                           GluIleSerSerCysThrValAspArgAspThrValCysGlyCysArg                              115120125                                                                     AAGAACCAGTACCGGCATTATTGGAGTGAAAACCTTTTCCAGTGCTTC432                           LysAsnGlnTyrArgHisTyrTrpSerGluAsnLeuPheGlnCysPhe                              130135140                                                                     AATTGCAGCCTCTGCCTCAATGGGACCGTGCACCTCTCCTGCCAGGAG480                           AsnCysSerLeuCysLeuAsnGlyThrValHisLeuSerCysGlnGlu                              145150155160                                                                  AAACAGAACACCGTGTGCACC501                                                      LysGlnAsnThrValCysThr                                                         165                                                                           (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 167 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      MetGlyLeuSerThrValProAspLeuLeuLeuProLeuValLeuLeu                              151015                                                                        GluLeuLeuValGlyIleTyrProSerGlyValIleGlyLeuValPro                              202530                                                                        HisLeuGlyAspArgGluLysArgAspSerValCysProGlnGlyLys                              354045                                                                        TyrIleHisProGlnAsnAsnSerIleCysCysThrLysCysHisLys                              505560                                                                        GlyThrTyrLeuTyrAsnAspCysProGlyProGlyGlnAspThrAsp                              65707580                                                                      CysArgGluCysGluSerGlySerPheThrAlaSerGluAsnHisLeu                              859095                                                                        ArgHisCysLeuSerCysSerLysCysArgLysGluMetGlyGlnVal                              100105110                                                                     GluIleSerSerCysThrValAspArgAspThrValCysGlyCysArg                              115120125                                                                     LysAsnGlnTyrArgHisTyrTrpSerGluAsnLeuPheGlnCysPhe                              130135140                                                                     AsnCysSerLeuCysLeuAsnGlyThrValHisLeuSerCysGlnGlu                              145150155160                                                                  LysGlnAsnThrValCysThr                                                         165                                                                           (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 372 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..372                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      GTGTGTCCCCAAGGAAAATATATCCACCCTCAAAATAATTCGATTTGC48                            ValCysProGlnGlyLysTyrIleHisProGlnAsnAsnSerIleCys                              151015                                                                        TGTACCAAGTGCCACAAAGGAACCTACTTGTACAATGACTGTCCAGGC96                            CysThrLysCysHisLysGlyThrTyrLeuTyrAsnAspCysProGly                              202530                                                                        CCGGGGCAGGATACGGACTGCAGGGAGTGTGAGAGCGGCTCCTTCACC144                           ProGlyGlnAspThrAspCysArgGluCysGluSerGlySerPheThr                              354045                                                                        GCTTCAGAAAACCACCTCAGACACTGCCTCAGCTGCTCCAAATGCCGA192                           AlaSerGluAsnHisLeuArgHisCysLeuSerCysSerLysCysArg                              505560                                                                        AAGGAAATGGGTCAGGTGGAGATCTCTTCTTGCACAGTGGACCGGGAC240                           LysGluMetGlyGlnValGluIleSerSerCysThrValAspArgAsp                              65707580                                                                      ACCGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGGAGTGAA288                           ThrValCysGlyCysArgLysAsnGlnTyrArgHisTyrTrpSerGlu                              859095                                                                        AACCTTTTCCAGTGCTTCAATTGCAGCCTCTGCCTCAATGGGACCGTG336                           AsnLeuPheGlnCysPheAsnCysSerLeuCysLeuAsnGlyThrVal                              100105110                                                                     CACCTCTCCTGCCAGGAGAAACAGAACACCGTGTGC372                                       HisLeuSerCysGlnGluLysGlnAsnThrValCys                                          115120                                                                        (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 124 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      ValCysProGlnGlyLysTyrIleHisProGlnAsnAsnSerIleCys                              151015                                                                        CysThrLysCysHisLysGlyThrTyrLeuTyrAsnAspCysProGly                              202530                                                                        ProGlyGlnAspThrAspCysArgGluCysGluSerGlySerPheThr                              354045                                                                        AlaSerGluAsnHisLeuArgHisCysLeuSerCysSerLysCysArg                              505560                                                                        LysGluMetGlyGlnValGluIleSerSerCysThrValAspArgAsp                              65707580                                                                      ThrValCysGlyCysArgLysAsnGlnTyrArgHisTyrTrpSerGlu                              859095                                                                        AsnLeuPheGlnCysPheAsnCysSerLeuCysLeuAsnGlyThrVal                              100105110                                                                     HisLeuSerCysGlnGluLysGlnAsnThrValCys                                          115120                                                                        (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      GluMetGlyGlnValGluIleSerSerThrValAspArgAspThrVal                              151015                                                                        CysGly                                                                        (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      AAGGAGATGGGCCAGGTTGAGATCTCTTCTACTGTTGACAATGACACTGTGTGTGGC57                   (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      GCTGCTCCAAATGCCGAAAG20                                                        (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      AGTTCAAGCTTTTACAGTGCCCTTAACATTCTAA34                                          (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      AspArgGluLysArg                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     GTTCTATCGATAAGAGGCCATAGCTGTCTGGC32                                            (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                     GCTCTCACACTCTCTCTTCTCCCTGTCCCCTAG33                                           (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     AGGGAGAAGAGAGAGTGTGAGAGCGGCTCCTTC33                                           (2) INFORMATION FOR SEQ ID NO: 13:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                     TGCATGGCAGGTACACACGGTGTCCCGGTCCAC33                                           (2) INFORMATION FOR SEQ ID NO: 14:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                     GACACCGTGTGTACCTGCCATGCAGGTTTCTTT33                                           (2) INFORMATION FOR SEQ ID NO: 15:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                     GGCCAAGCTTCAGGTGCACACGGTGTTCTG30                                              (2) INFORMATION FOR SEQ ID NO: 16:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                     GCTGCTCCAAATGCCGAAAG20                                                        (2) INFORMATION FOR SEQ ID NO: 17:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:                                     AGTTCAAGCTTTACAGTGCCCTTAACATTCTAA33                                           (2) INFORMATION FOR SEQ ID NO: 18:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:                                     CGCAGAATTCCCCGCAGCCATGGCGCCCGTCGCC34                                          (2) INFORMATION FOR SEQ ID NO: 19:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:                                     GTAAGGATCCTATCGCCAGTGCTCCCTTCAGCT33                                           (2) INFORMATION FOR SEQ ID NO: 20:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:                                     ACACGACTTCATCCACGGATA21                                                       (2) INFORMATION FOR SEQ ID NO: 21:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:                                     ACGTTCTAGACTAGTCGCCAGTGCTCCCTTCAGCTG36                                        (2) INFORMATION FOR SEQ ID NO: 22:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:                                     CAGAACCGCATCTGCACCTGC21                                                       (2) INFORMATION FOR SEQ ID NO: 23:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:                                     ACGTTCTAGACTTGCACACCACGTCTGATGTTTC34                                          (2) INFORMATION FOR SEQ ID NO: 24:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2062 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 155..1519                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:                                     ACCAGTGATCTCTATGCCCGAGTCTCAACCCTCAACTGTCACCCCAAGGCACTTGGGACG60                TCCTGGACAGACCGAGTCCCGGGAAGCCCCAGCACTGCCGCTGCCACACTGCCCTGAGCC120               CAAATGGGGGAGTGAGAGGCCATAGCTGTCTGGCATGGGCCTCTCCACCGTG172                       MetGlyLeuSerThrVal                                                            15                                                                            CCTGACCTGCTGCTGCCGCTGGTGCTCCTGGAGCTGTTGGTGGGAATA220                           ProAspLeuLeuLeuProLeuValLeuLeuGluLeuLeuValGlyIle                              101520                                                                        TACCCCTCAGGGGTTATTGGACTGGTCCCTCACCTAGGGGACAGGGAG268                           TyrProSerGlyValIleGlyLeuValProHisLeuGlyAspArgGlu                              253035                                                                        AAGAGAGATAGTGTGTGTCCCCAAGGAAAATATATCCACCCTCAAAAT316                           LysArgAspSerValCysProGlnGlyLysTyrIleHisProGlnAsn                              404550                                                                        AATTCGATTTGCTGTACCAAGTGCCACAAAGGAACCTACTTGTACAAT364                           AsnSerIleCysCysThrLysCysHisLysGlyThrTyrLeuTyrAsn                              55606570                                                                      GACTGTCCAGGCCCGGGGCAGGATACGGACTGCAGGGAGTGTGAGAGC412                           AspCysProGlyProGlyGlnAspThrAspCysArgGluCysGluSer                              758085                                                                        GGCTCCTTCACCGCTTCAGAAAACCACCTCAGACACTGCCTCAGCTGC460                           GlySerPheThrAlaSerGluAsnHisLeuArgHisCysLeuSerCys                              9095100                                                                       TCCAAATGCCGAAAGGAAATGGGTCAGGTGGAGATCTCTTCTTGCACA508                           SerLysCysArgLysGluMetGlyGlnValGluIleSerSerCysThr                              105110115                                                                     GTGGACCGGGACACCGTGTGTGGCTGCAGGAAGAACCAGTACCGGCAT556                           ValAspArgAspThrValCysGlyCysArgLysAsnGlnTyrArgHis                              120125130                                                                     TATTGGAGTGAAAACCTTTTCCAGTGCTTCAATTGCAGCCTCTGCCTC604                           TyrTrpSerGluAsnLeuPheGlnCysPheAsnCysSerLeuCysLeu                              135140145150                                                                  AATGGGACCGTGCACCTCTCCTGCCAGGAGAAACAGAACACCGTGTGC652                           AsnGlyThrValHisLeuSerCysGlnGluLysGlnAsnThrValCys                              155160165                                                                     ACCTGCCATGCAGGTTTCTTTCTAAGAGAAAACGAGTGTGTCTCCTGT700                           ThrCysHisAlaGlyPhePheLeuArgGluAsnGluCysValSerCys                              170175180                                                                     AGTAACTGTAAGAAAAGCCTGGAGTGCACGAAGTTGTGCCTACCCCAG748                           SerAsnCysLysLysSerLeuGluCysThrLysLeuCysLeuProGln                              185190195                                                                     ATTGAGAATGTTAAGGGCACTGAGGACTCAGGCACCACAGTGCTGTTG796                           IleGluAsnValLysGlyThrGluAspSerGlyThrThrValLeuLeu                              200205210                                                                     CCCCTGGTCATTTTCTTTGGTCTTTGCCTTTTATCCCTCCTCTTCATT844                           ProLeuValIlePhePheGlyLeuCysLeuLeuSerLeuLeuPheIle                              215220225230                                                                  GGTTTAATGTATCGCTACCAACGGTGGAAGTCCAAGCTCTACTCCATT892                           GlyLeuMetTyrArgTyrGlnArgTrpLysSerLysLeuTyrSerIle                              235240245                                                                     GTTTGTGGGAAATCGACACCTGAAAAAGAGGGGGAGCTTGAAGGAACT940                           ValCysGlyLysSerThrProGluLysGluGlyGluLeuGluGlyThr                              250255260                                                                     ACTACTAAGCCCCTGGCCCCAAACCCAAGCTTCAGTCCCACTCCAGGC988                           ThrThrLysProLeuAlaProAsnProSerPheSerProThrProGly                              265270275                                                                     TTCACCCCCACCCTGGGCTTCAGTCCCGTGCCCAGTTCCACCTTCACC1036                          PheThrProThrLeuGlyPheSerProValProSerSerThrPheThr                              280285290                                                                     TCCAGCTCCACCTATACCCCCGGTGACTGTCCCAACTTTGCGGCTCCC1084                          SerSerSerThrTyrThrProGlyAspCysProAsnPheAlaAlaPro                              295300305310                                                                  CGCAGAGAGGTGGCACCACCCTATCAGGGGGCTGACCCCATCCTTGCG1132                          ArgArgGluValAlaProProTyrGlnGlyAlaAspProIleLeuAla                              315320325                                                                     ACAGCCCTCGCCTCCGACCCCATCCCCAACCCCCTTCAGAAGTGGGAG1180                          ThrAlaLeuAlaSerAspProIleProAsnProLeuGlnLysTrpGlu                              330335340                                                                     GACAGTGCCCACAAGCCACAGAGCCTAGACACTGATGACCCCCGGACG1228                          AspSerAlaHisLysProGlnSerLeuAspThrAspAspProArgThr                              345350355                                                                     CTGTACGCCGTGGTGGAGAACGTGCCCCCGTTGCGCTGGAAGGAATTC1276                          LeuTyrAlaValValGluAsnValProProLeuArgTrpLysGluPhe                              360365370                                                                     GTGCGGCGCCTAGGGCTGAGCGACCACGAGATCGATCGGCTGGAGCTG1324                          ValArgArgLeuGlyLeuSerAspHisGluIleAspArgLeuGluLeu                              375380385390                                                                  CAGAACGGGCGCTGCCTGCGCGAGGCGCAATACAGCATGCTGGCGACC1372                          GlnAsnGlyArgCysLeuArgGluAlaGlnTyrSerMetLeuAlaThr                              395400405                                                                     TGGAGGCGGCGCACGCCGCGGCGCGAGGCCACGCTGGAGCTGCTGGGA1420                          TrpArgArgArgThrProArgArgGluAlaThrLeuGluLeuLeuGly                              410415420                                                                     CGCGTGCTCCGCGACATGGACCTGCTGGGCTGCCTGGAGGACATCGAG1468                          ArgValLeuArgAspMetAspLeuLeuGlyCysLeuGluAspIleGlu                              425430435                                                                     GAGGCGCTTTGCGGCCCCGCCGCGCTCCCGCCCGCGCCCAGTCTTCTC1516                          GluAlaLeuCysGlyProAlaAlaLeuProProAlaProSerLeuLeu                              440445450                                                                     AGATGAGGCTGCGCCCTGCGGGCAGCTCTAAGGACCGTCCTCGCAGATCGCCT1569                     Arg                                                                           455                                                                           TCCAACCCCACTTTTTTCTGGAAAGGAGGGGTCCTGCAGGGGCAAGCAGGAGCTAGCAGC1629              CGCCTACTTGGTGCTAACCCCTCGATGTACATAGCTTTTCTCAGCTGCCTGCGCGCCGCC1689              GACAGTCAGCGCTGTGCGCGCGGAGAGAGGTGCGCCGTGGGCTCAAGAGCCTGAGTGGGT1749              GGTTTGCGAGGATGAGGGACGCTATGCCTCATGCCCGTTTTGGGTGTCCTCACCAGCAAG1809              GCTGCTCGGGGGCCCCTGGTTCGTCCCTGAGCCTTTTTCACAGTGCATAAGCAGTTTTTT1869              TTGTTTTTGTTTTGTTTTGTTTTGTTTTTAAATCAATCATGTTACACTAATAGAAACTTG1929              GCACTCCTGTGCCCTCTGCCTGGACAAGCACATAGCAAGCTGAACTGTCCTAAGGCAGGG1989              GCGAGCACGGAACAATGGGGCCTTCAGCTGGAGCTGTGGACTTTTGTACATACACTAAAA2049              TTCTGAAGTTAAG2062                                                             (2) INFORMATION FOR SEQ ID NO: 25:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 455 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:                                     MetGlyLeuSerThrValProAspLeuLeuLeuProLeuValLeuLeu                              151015                                                                        GluLeuLeuValGlyIleTyrProSerGlyValIleGlyLeuValPro                              202530                                                                        HisLeuGlyAspArgGluLysArgAspSerValCysProGlnGlyLys                              354045                                                                        TyrIleHisProGlnAsnAsnSerIleCysCysThrLysCysHisLys                              505560                                                                        GlyThrTyrLeuTyrAsnAspCysProGlyProGlyGlnAspThrAsp                              65707580                                                                      CysArgGluCysGluSerGlySerPheThrAlaSerGluAsnHisLeu                              859095                                                                        ArgHisCysLeuSerCysSerLysCysArgLysGluMetGlyGlnVal                              100105110                                                                     GluIleSerSerCysThrValAspArgAspThrValCysGlyCysArg                              115120125                                                                     LysAsnGlnTyrArgHisTyrTrpSerGluAsnLeuPheGlnCysPhe                              130135140                                                                     AsnCysSerLeuCysLeuAsnGlyThrValHisLeuSerCysGlnGlu                              145150155160                                                                  LysGlnAsnThrValCysThrCysHisAlaGlyPhePheLeuArgGlu                              165170175                                                                     AsnGluCysValSerCysSerAsnCysLysLysSerLeuGluCysThr                              180185190                                                                     LysLeuCysLeuProGlnIleGluAsnValLysGlyThrGluAspSer                              195200205                                                                     GlyThrThrValLeuLeuProLeuValIlePhePheGlyLeuCysLeu                              210215220                                                                     LeuSerLeuLeuPheIleGlyLeuMetTyrArgTyrGlnArgTrpLys                              225230235240                                                                  SerLysLeuTyrSerIleValCysGlyLysSerThrProGluLysGlu                              245250255                                                                     GlyGluLeuGluGlyThrThrThrLysProLeuAlaProAsnProSer                              260265270                                                                     PheSerProThrProGlyPheThrProThrLeuGlyPheSerProVal                              275280285                                                                     ProSerSerThrPheThrSerSerSerThrTyrThrProGlyAspCys                              290295300                                                                     ProAsnPheAlaAlaProArgArgGluValAlaProProTyrGlnGly                              305310315320                                                                  AlaAspProIleLeuAlaThrAlaLeuAlaSerAspProIleProAsn                              325330335                                                                     ProLeuGlnLysTrpGluAspSerAlaHisLysProGlnSerLeuAsp                              340345350                                                                     ThrAspAspProArgThrLeuTyrAlaValValGluAsnValProPro                              355360365                                                                     LeuArgTrpLysGluPheValArgArgLeuGlyLeuSerAspHisGlu                              370375380                                                                     IleAspArgLeuGluLeuGlnAsnGlyArgCysLeuArgGluAlaGln                              385390395400                                                                  TyrSerMetLeuAlaThrTrpArgArgArgThrProArgArgGluAla                              405410415                                                                     ThrLeuGluLeuLeuGlyArgValLeuArgAspMetAspLeuLeuGly                              420425430                                                                     CysLeuGluAspIleGluGluAlaLeuCysGlyProAlaAlaLeuPro                              435440445                                                                     ProAlaProSerLeuLeuArg                                                         450455                                                                        (2) INFORMATION FOR SEQ ID NO: 26:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:                                     ValCysProGlnGlyLysTyrIleHisProGlnAsnAsnSerIleCys                              151015                                                                        CysThrLysCysHisLysGlyThrTyrLeuTyrAsnAspCysProGly                              202530                                                                        ProGlyGlnAspThrAspCysArg                                                      3540                                                                          (2) INFORMATION FOR SEQ ID NO: 27:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:                                     ThrCysArgLeuArgGluTyrTyrAspGlnThrAlaGlnMetCysCys                              151015                                                                        SerLysCysSerProGlyGlnHisAlaLysValPheCysThrLysThr                              202530                                                                        SerAspThrValCysAsp                                                            35                                                                            (2) INFORMATION FOR SEQ ID NO: 28:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:                                     ThrCysSerThrGlyLeuTyrThrHisSerGlyGluCysCysLysAla                              151015                                                                        CysAsnLeuGlyGluGlyValAlaGlnProCysGlyAlaAsnGlnThr                              202530                                                                        ValCysGlu                                                                     35                                                                            (2) INFORMATION FOR SEQ ID NO: 29:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:                                     AlaCysArgGluLysGlnTyrLeuIleAsnSerGlnCysCysSerLeu                              151015                                                                        CysGlnProGlyGlnLysLeuValSerAspCysThrGluPheThrGlu                              202530                                                                        ThrGluCysLeu                                                                  35                                                                            (2) INFORMATION FOR SEQ ID NO: 30:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:                                     AsnCysValLysAspThrTyrProSerGlyHisLysCysCysArgGlu                              151015                                                                        CysGlnProGlyHisGlyMetValSerArgCysAspHisThrArgAsp                              202530                                                                        ThrValCysHis                                                                  35                                                                            (2) INFORMATION FOR SEQ ID NO: 31:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:                                     GluCysGluSerGlySerPheThrAlaSerGluAsnHisLeuArgHis                              151015                                                                        CysLeuSerCysSerLysCysArgLysGluMetGlyGlnValGluIle                              202530                                                                        SerSerCysThrValAspArgAspThrValCys                                             3540                                                                          (2) INFORMATION FOR SEQ ID NO: 32:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:                                     SerCysGluAspSerThrTyrThrGlnLeuTrpAsnTrpValProGlu                              151015                                                                        CysLeuSerCysGlySerArgCysSerSerAspGlnValGluThrGln                              202530                                                                        AlaCysThrArgGluGlnAsnArgIleCys                                                3540                                                                          (2) INFORMATION FOR SEQ ID NO: 33:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:                                     ProCysLeuAspAsnValThrPheSerAspValValSerAlaThrGlu                              151015                                                                        ProCysLysProCysThrGluCysLeuGlyLeuGlnSerMetSerAla                              202530                                                                        ProCysValGluAlaAspAspAlaValCys                                                3540                                                                          (2) INFORMATION FOR SEQ ID NO: 34:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:                                     ProCysGlyGluSerGluPheLeuAspThrTrpAsnArgGluThrHis                              151015                                                                        CysHisGlnHisLysTyrCysAspProAsnLeuGlyLeuArgValGln                              202530                                                                        GlnLysGlyThrSerGluThrAspThrIleCys                                             3540                                                                          (2) INFORMATION FOR SEQ ID NO: 35:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:                                     ProCysGluProGlyPheTyrAsnGluAlaValAsnTyrAspThrCys                              151015                                                                        LysGlnCysThrGlnCysAsnHisArgSerGlySerGluLeuLysGln                              202530                                                                        AsnCysThrProThrGluAspThrValCys                                                3540                                                                          (2) INFORMATION FOR SEQ ID NO: 36:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:                                     GlyCysArgLysAsnGlnTyrArgHisTyrTrpSerGluAsnLeuPhe                              151015                                                                        GlnCysPheAsnCysSerLeuCysLeuAsnGlyThrValHisLeuSer                              202530                                                                        CysGlnGluLysGlnAsnThrValCys                                                   3540                                                                          (2) INFORMATION FOR SEQ ID NO: 37:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:                                     ThrCysArgProGlyTrpTyrCysAlaLeuSerLysGlnGluGlyCys                              151015                                                                        ArgLeuCysAlaProLeuArgLysCysArgProGlyPheGlyValAla                              202530                                                                        ArgProGlyThrGluThrSerAspValValCysLys                                          3540                                                                          (2) INFORMATION FOR SEQ ID NO: 38:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:                                     ArgCysAlaTyrGlyTyrTyrGlnAspGluGluThrGlyHisCysGlu                              151015                                                                        AlaCysSerValCysGluValGlySerGlyLeuValPheSerCysGln                              202530                                                                        AspLysGlnAsnThrValCysGlu                                                      3540                                                                          (2) INFORMATION FOR SEQ ID NO: 39:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:                                     ThrCysGluGluGlyTrpHisCysThrSerGluAlaCysGluSerCys                              151015                                                                        ValLeuHisArgSerCysSerProGlyPheGlyValLysGlnIleAla                              202530                                                                        ThrGlyValSerAspThrIleCysGlu                                                   3540                                                                          (2) INFORMATION FOR SEQ ID NO: 40:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:                                     ThrCysHisAlaGlyPhePheLeuArgGluAsnGluCysValSerCys                              151015                                                                        SerAsnCysLysLysSerLeuGluCysThrLysLeuCysLeuProGln                              202530                                                                        IleGluAsnValLysGlyThr                                                         35                                                                            (2) INFORMATION FOR SEQ ID NO: 41:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:                                     ProCysAlaProGlyThrPheSerAsnThrThrSerSerThrAspIle                              151015                                                                        CysArgProHisGlnIleCysAsnValValAlaIleProGlyAsnAla                              202530                                                                        SerMetAspAlaValCysThr                                                         35                                                                            (2) INFORMATION FOR SEQ ID NO: 42:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:                                     GluCysProGluGlyThrTyrSerAspGluAlaAsnHisValAspPro                              151015                                                                        CysLeuProCysThrValCysGluAspThrGluArgGlnLeuArgGlu                              202530                                                                        CysThrProTrpAlaAspAlaGluCysGlu                                                3540                                                                          (2) INFORMATION FOR SEQ ID NO: 43:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:                                     ProCysProValGlyPhePheSerAsnValSerSerAlaPheGluLys                              151015                                                                        CysHisProTrpThrSerCysGluThrLysAspLeuValValGlnGln                              202530                                                                        AlaGlyThrAsnLysThrAspValValCysGly                                             3540                                                                          (2) INFORMATION FOR SEQ ID NO: 44:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:                                     ProCysProProGlyHisPheSerProGlySerAsnGlnAlaCysLys                              151015                                                                        ProTrpThrAsnCysThrLeuSerGlyLysGlnIleArgHisProAla                              202530                                                                        SerAsnSerLeuAspThrValCysGlu                                                   3540                                                                          (2) INFORMATION FOR SEQ ID NO: 45:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:                                     TGTCTGGCATGG12                                                                (2) INFORMATION FOR SEQ ID NO: 46:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:                                     CCCCAGATTTAG12                                                                (2) INFORMATION FOR SEQ ID NO: 47:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 600 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..597                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:                                     ATGGGCCTCTCCACCGTGCCTGACCTGCTGCTGCCGCTGGTGCTCCTG48                            MetGlyLeuSerThrValProAspLeuLeuLeuProLeuValLeuLeu                              151015                                                                        GAGCTGTTGGTGGGAATATACCCCTCAGGGGTTATTGGACTGGTCCCT96                            GluLeuLeuValGlyIleTyrProSerGlyValIleGlyLeuValPro                              202530                                                                        CACCTAGGGGACAGGGAGAAGAGAGATAGTGTGTGTCCCACAGGAAAA144                           HisLeuGlyAspArgGluLysArgAspSerValCysProThrGlyLys                              354045                                                                        TATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGTGCCACAAA192                           TyrIleHisProGlnAsnAsnSerIleCysCysThrLysCysHisLys                              505560                                                                        GGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGAC240                           GlyThrTyrLeuTyrAsnAspCysProGlyProGlyGlnAspThrAsp                              65707580                                                                      TGCAGGGAGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTC288                           CysArgGluCysGluSerGlySerPheThrAlaSerGluAsnHisLeu                              859095                                                                        AGACACTGCCTCAGCTGCTCCAAATGCCGAAAGGAAATGGGTCAGGTG336                           ArgHisCysLeuSerCysSerLysCysArgLysGluMetGlyGlnVal                              100105110                                                                     GAGATCTCTTCTTGCACAGTGGACCGGGACACCGTGTGTGGCTGCAGG384                           GluIleSerSerCysThrValAspArgAspThrValCysGlyCysArg                              115120125                                                                     AAGAACCAGTACCGGCATTATTGGAGTGAAAACCTTTTCCAGTGCTTC432                           LysAsnGlnTyrArgHisTyrTrpSerGluAsnLeuPheGlnCysPhe                              130135140                                                                     AATTGCAGCCTCTGCCTCAATGGGACCGTGCACCTCTCCTGCCAGGAG480                           AsnCysSerLeuCysLeuAsnGlyThrValHisLeuSerCysGlnGlu                              145150155160                                                                  AAACAGAACACCGTGTGCACCTGCCATGCAGGTTTCTTTCTAAGAGAA528                           LysGlnAsnThrValCysThrCysHisAlaGlyPhePheLeuArgGlu                              165170175                                                                     AACGAGTGTGTCTCCTGTAGTAACTGTAAGAAAAGCCTGGAGTGCACG576                           AsnGluCysValSerCysSerAsnCysLysLysSerLeuGluCysThr                              180185190                                                                     AAGTTGTGCCTACCCCAGATTTAG600                                                   LysLeuCysLeuProGlnIle                                                         195200                                                                        (2) INFORMATION FOR SEQ ID NO: 48:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 199 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:                                     MetGlyLeuSerThrValProAspLeuLeuLeuProLeuValLeuLeu                              151015                                                                        GluLeuLeuValGlyIleTyrProSerGlyValIleGlyLeuValPro                              202530                                                                        HisLeuGlyAspArgGluLysArgAspSerValCysProThrGlyLys                              354045                                                                        TyrIleHisProGlnAsnAsnSerIleCysCysThrLysCysHisLys                              505560                                                                        GlyThrTyrLeuTyrAsnAspCysProGlyProGlyGlnAspThrAsp                              65707580                                                                      CysArgGluCysGluSerGlySerPheThrAlaSerGluAsnHisLeu                              859095                                                                        ArgHisCysLeuSerCysSerLysCysArgLysGluMetGlyGlnVal                              100105110                                                                     GluIleSerSerCysThrValAspArgAspThrValCysGlyCysArg                              115120125                                                                     LysAsnGlnTyrArgHisTyrTrpSerGluAsnLeuPheGlnCysPhe                              130135140                                                                     AsnCysSerLeuCysLeuAsnGlyThrValHisLeuSerCysGlnGlu                              145150155160                                                                  LysGlnAsnThrValCysThrCysHisAlaGlyPhePheLeuArgGlu                              165170175                                                                     AsnGluCysValSerCysSerAsnCysLysLysSerLeuGluCysThr                              180185190                                                                     LysLeuCysLeuProGlnIle                                                         195                                                                           (2) INFORMATION FOR SEQ ID NO: 49:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 474 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..471                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:                                     ATGGGCCTCTCCACCGTGCCTGACCTGCTGCTGCCGCTGGTGCTCCTG48                            MetGlyLeuSerThrValProAspLeuLeuLeuProLeuValLeuLeu                              151015                                                                        GAGCTGTTGGTGGGAATATACCCCTCAGGGGTTATTGGACTGGTCCCT96                            GluLeuLeuValGlyIleTyrProSerGlyValIleGlyLeuValPro                              202530                                                                        CACCTAGGGGACAGGGAGAAGAGAGAGTGTGAGAGCGGCTCCTTCACC144                           HisLeuGlyAspArgGluLysArgGluCysGluSerGlySerPheThr                              354045                                                                        GCTTCAGAAAACCACCTCAGACACTGCCTCAGCTGCTCCAAATGCCGA192                           AlaSerGluAsnHisLeuArgHisCysLeuSerCysSerLysCysArg                              505560                                                                        AAGGAAATGGGTCAGGTGGAGATCTCTTCTTGCACAGTGGACCGGGAC240                           LysGluMetGlyGlnValGluIleSerSerCysThrValAspArgAsp                              65707580                                                                      ACCGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGGAGTGAA288                           ThrValCysGlyCysArgLysAsnGlnTyrArgHisTyrTrpSerGlu                              859095                                                                        AACCTTTTCCAGTGCTTCAATTGCAGCCTCTGCCTCAATGGGACCGTG336                           AsnLeuPheGlnCysPheAsnCysSerLeuCysLeuAsnGlyThrVal                              100105110                                                                     CACCTCTCCTGCCAGGAGAAACAGAACACCGTGTGCACCTGCCATGCA384                           HisLeuSerCysGlnGluLysGlnAsnThrValCysThrCysHisAla                              115120125                                                                     GGTTTCTTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTAACTGTAAG432                           GlyPhePheLeuArgGluAsnGluCysValSerCysSerAsnCysLys                              130135140                                                                     AAAAGCCTGGAGTGCACGAAGTTGTGCCTACCCCAGATTTAG474                                 LysSerLeuGluCysThrLysLeuCysLeuProGlnIle                                       145150155                                                                     (2) INFORMATION FOR SEQ ID NO: 50:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 157 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:                                     MetGlyLeuSerThrValProAspLeuLeuLeuProLeuValLeuLeu                              151015                                                                        GluLeuLeuValGlyIleTyrProSerGlyValIleGlyLeuValPro                              202530                                                                        HisLeuGlyAspArgGluLysArgGluCysGluSerGlySerPheThr                              354045                                                                        AlaSerGluAsnHisLeuArgHisCysLeuSerCysSerLysCysArg                              505560                                                                        LysGluMetGlyGlnValGluIleSerSerCysThrValAspArgAsp                              65707580                                                                      ThrValCysGlyCysArgLysAsnGlnTyrArgHisTyrTrpSerGlu                              859095                                                                        AsnLeuPheGlnCysPheAsnCysSerLeuCysLeuAsnGlyThrVal                              100105110                                                                     HisLeuSerCysGlnGluLysGlnAsnThrValCysThrCysHisAla                              115120125                                                                     GlyPhePheLeuArgGluAsnGluCysValSerCysSerAsnCysLys                              130135140                                                                     LysSerLeuGluCysThrLysLeuCysLeuProGlnIle                                       145150155                                                                     (2) INFORMATION FOR SEQ ID NO: 51:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 462 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..459                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:                                     ATGGGCCTCTCCACCGTGCCTGACCTGCTGCTGCCGCTGGTGCTCCTG48                            MetGlyLeuSerThrValProAspLeuLeuLeuProLeuValLeuLeu                              151015                                                                        GAGCTGTTGGTGGGAATATACCCCTCAGGGGTTATTGGACTGGTCCCT96                            GluLeuLeuValGlyIleTyrProSerGlyValIleGlyLeuValPro                              202530                                                                        CACCTAGGGGACAGGGAGAAGAGAGATAGTGTGTGTCCCCAAGGAAAA144                           HisLeuGlyAspArgGluLysArgAspSerValCysProGlnGlyLys                              354045                                                                        TATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGTGCCACAAA192                           TyrIleHisProGlnAsnAsnSerIleCysCysThrLysCysHisLys                              505560                                                                        GGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGAC240                           GlyThrTyrLeuTyrAsnAspCysProGlyProGlyGlnAspThrAsp                              65707580                                                                      TGCAGGAAGAACCAGTACCGGCATTATTGGAGTGAAAACCTTTTCCAG288                           CysArgLysAsnGlnTyrArgHisTyrTrpSerGluAsnLeuPheGln                              859095                                                                        TGCTTCAATTGCAGCCTCTGCCTCAATGGGACCGTGCACCTCTCCTGC336                           CysPheAsnCysSerLeuCysLeuAsnGlyThrValHisLeuSerCys                              100105110                                                                     CAGGAGAAACAGAACACCGTGTGCACCTGCCATGCAGGTTTCTTTCTA384                           GlnGluLysGlnAsnThrValCysThrCysHisAlaGlyPhePheLeu                              115120125                                                                     AGAGAAAACGAGTGTGTCTCCTGTAGTAACTGTAAGAAAAGCCTGGAG432                           ArgGluAsnGluCysValSerCysSerAsnCysLysLysSerLeuGlu                              130135140                                                                     TGCACGAAGTTGTGCCTACCCCAGATTTAG462                                             CysThrLysLeuCysLeuProGlnIle                                                   145150                                                                        (2) INFORMATION FOR SEQ ID NO: 52:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 153 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:                                     MetGlyLeuSerThrValProAspLeuLeuLeuProLeuValLeuLeu                              151015                                                                        GluLeuLeuValGlyIleTyrProSerGlyValIleGlyLeuValPro                              202530                                                                        HisLeuGlyAspArgGluLysArgAspSerValCysProGlnGlyLys                              354045                                                                        TyrIleHisProGlnAsnAsnSerIleCysCysThrLysCysHisLys                              505560                                                                        GlyThrTyrLeuTyrAsnAspCysProGlyProGlyGlnAspThrAsp                              65707580                                                                      CysArgLysAsnGlnTyrArgHisTyrTrpSerGluAsnLeuPheGln                              859095                                                                        CysPheAsnCysSerLeuCysLeuAsnGlyThrValHisLeuSerCys                              100105110                                                                     GlnGluLysGlnAsnThrValCysThrCysHisAlaGlyPhePheLeu                              115120125                                                                     ArgGluAsnGluCysValSerCysSerAsnCysLysLysSerLeuGlu                              130135140                                                                     CysThrLysLeuCysLeuProGlnIle                                                   145150                                                                        (2) INFORMATION FOR SEQ ID NO: 53:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 477 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..474                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:                                     ATGGGCCTCTCCACCGTGCCTGACCTGCTGCTGCCGCTGGTGCTCCTG48                            MetGlyLeuSerThrValProAspLeuLeuLeuProLeuValLeuLeu                              151015                                                                        GAGCTGTTGGTGGGAATATACCCCTCAGGGGTTATTGGACTGGTCCCT96                            GluLeuLeuValGlyIleTyrProSerGlyValIleGlyLeuValPro                              202530                                                                        CACCTAGGGGACAGGGAGAAGAGAGATAGTGTGTGTCCCCAAGGAAAA144                           HisLeuGlyAspArgGluLysArgAspSerValCysProGlnGlyLys                              354045                                                                        TATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGTGCCACAAA192                           TyrIleHisProGlnAsnAsnSerIleCysCysThrLysCysHisLys                              505560                                                                        GGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGAC240                           GlyThrTyrLeuTyrAsnAspCysProGlyProGlyGlnAspThrAsp                              65707580                                                                      TGCAGGGAGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTC288                           CysArgGluCysGluSerGlySerPheThrAlaSerGluAsnHisLeu                              859095                                                                        AGACACTGCCTCAGCTGCTCCAAATGCCGAAAGGAAATGGGTCAGGTG336                           ArgHisCysLeuSerCysSerLysCysArgLysGluMetGlyGlnVal                              100105110                                                                     GAGATCTCTTCTTGCACAGTGGACCGGGACACCGTGTGTACCTGCCAT384                           GluIleSerSerCysThrValAspArgAspThrValCysThrCysHis                              115120125                                                                     GCAGGTTTCTTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTAACTGT432                           AlaGlyPhePheLeuArgGluAsnGluCysValSerCysSerAsnCys                              130135140                                                                     AAGAAAAGCCTGGAGTGCACGAAGTTGTGCCTACCCCAGATTTAG477                              LysLysSerLeuGluCysThrLysLeuCysLeuProGlnIle                                    145150155                                                                     (2) INFORMATION FOR SEQ ID NO: 54:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 158 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:                                     MetGlyLeuSerThrValProAspLeuLeuLeuProLeuValLeuLeu                              151015                                                                        GluLeuLeuValGlyIleTyrProSerGlyValIleGlyLeuValPro                              202530                                                                        HisLeuGlyAspArgGluLysArgAspSerValCysProGlnGlyLys                              354045                                                                        TyrIleHisProGlnAsnAsnSerIleCysCysThrLysCysHisLys                              505560                                                                        GlyThrTyrLeuTyrAsnAspCysProGlyProGlyGlnAspThrAsp                              65707580                                                                      CysArgGluCysGluSerGlySerPheThrAlaSerGluAsnHisLeu                              859095                                                                        ArgHisCysLeuSerCysSerLysCysArgLysGluMetGlyGlnVal                              100105110                                                                     GluIleSerSerCysThrValAspArgAspThrValCysThrCysHis                              115120125                                                                     AlaGlyPhePheLeuArgGluAsnGluCysValSerCysSerAsnCys                              130135140                                                                     LysLysSerLeuGluCysThrLysLeuCysLeuProGlnIle                                    145150155                                                                     (2) INFORMATION FOR SEQ ID NO: 55:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:                                     GTGTGCACCTGA12                                                                (2) INFORMATION FOR SEQ ID NO: 56:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 504 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..501                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:                                     ATGGGCCTCTCCACCGTGCCTGACCTGCTGCTGCCGCTGGTGCTCCTG48                            MetGlyLeuSerThrValProAspLeuLeuLeuProLeuValLeuLeu                              151015                                                                        GAGCTGTTGGTGGGAATATACCCCTCAGGGGTTATTGGACTGGTCCCT96                            GluLeuLeuValGlyIleTyrProSerGlyValIleGlyLeuValPro                              202530                                                                        CACCTAGGGGACAGGGAGAAGAGAGATAGTGTGTGTCCCCAAGGAAAA144                           HisLeuGlyAspArgGluLysArgAspSerValCysProGlnGlyLys                              354045                                                                        TATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGTGCCACAAA192                           TyrIleHisProGlnAsnAsnSerIleCysCysThrLysCysHisLys                              505560                                                                        GGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGAC240                           GlyThrTyrLeuTyrAsnAspCysProGlyProGlyGlnAspThrAsp                              65707580                                                                      TGCAGGGAGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTC288                           CysArgGluCysGluSerGlySerPheThrAlaSerGluAsnHisLeu                              859095                                                                        AGACACTGCCTCAGCTGCTCCAAATGCCGAAAGGAAATGGGTCAGGTG336                           ArgHisCysLeuSerCysSerLysCysArgLysGluMetGlyGlnVal                              100105110                                                                     GAGATCTCTTCTTGCACAGTGGACCGGGACACCGTGTGTGGCTGCAGG384                           GluIleSerSerCysThrValAspArgAspThrValCysGlyCysArg                              115120125                                                                     AAGAACCAGTACCGGCATTATTGGAGTGAAAACCTTTTCCAGTGCTTC432                           LysAsnGlnTyrArgHisTyrTrpSerGluAsnLeuPheGlnCysPhe                              130135140                                                                     AATTGCAGCCTCTGCCTCAATGGGACCGTGCACCTCTCCTGCCAGGAG480                           AsnCysSerLeuCysLeuAsnGlyThrValHisLeuSerCysGlnGlu                              145150155160                                                                  AAACAGAACACCGTGTGCACCTGA504                                                   LysGlnAsnThrValCysThr                                                         165                                                                           (2) INFORMATION FOR SEQ ID NO: 57:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 167 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:                                     MetGlyLeuSerThrValProAspLeuLeuLeuProLeuValLeuLeu                              151015                                                                        GluLeuLeuValGlyIleTyrProSerGlyValIleGlyLeuValPro                              202530                                                                        HisLeuGlyAspArgGluLysArgAspSerValCysProGlnGlyLys                              354045                                                                        TyrIleHisProGlnAsnAsnSerIleCysCysThrLysCysHisLys                              505560                                                                        GlyThrTyrLeuTyrAsnAspCysProGlyProGlyGlnAspThrAsp                              65707580                                                                      CysArgGluCysGluSerGlySerPheThrAlaSerGluAsnHisLeu                              859095                                                                        ArgHisCysLeuSerCysSerLysCysArgLysGluMetGlyGlnVal                              100105110                                                                     GluIleSerSerCysThrValAspArgAspThrValCysGlyCysArg                              115120125                                                                     LysAsnGlnTyrArgHisTyrTrpSerGluAsnLeuPheGlnCysPhe                              130135140                                                                     AsnCysSerLeuCysLeuAsnGlyThrValHisLeuSerCysGlnGlu                              145150155160                                                                  LysGlnAsnThrValCysThr                                                         165                                                                           __________________________________________________________________________

We claim:
 1. A polypeptide which is capable of binding human TNFα andwhich has the first three cysteine-rich subdomains, but not the fourthcysteine-rich subdomain, of the extracellular binding domain of areceptor selected from the group consisting of the 55 kD and 75 kDreceptors for human TNFα.
 2. A polypeptide according to claim 1, whichhas the amino acid sequence of SEQ ID NO:
 2. 3. A polypeptide accordingto claim 1, which has the amino acid sequence of SEQ ID NO:
 4. 4. Aprocess for the preparation of a polypeptide which is capable of bindinghuman TNFα and which has the first three cysteine-rich subdomains butnot the fourth cysteine-rich subdomain of the extracellular bindingdomain of a receptor selected from the group consisting of the 55 kD and75 kD receptors for human TNFα, which process comprises(i) culturing ahost transformed with a vector encoding said polypeptide under suchconditions that said polypeptide is expressed; and (ii) isolating saidpolypeptide.
 5. A process according to claim 4, wherein said polypeptidehas the amino acid sequence of SEQ ID NO:
 2. 6. A process according toclaim 4, wherein said polypeptide has the amino acid sequence of SEQ IDNO:
 4. 7. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier or diluent and, as an active principle, a polypeptidewhich is capable of binding human TNFα and which has the first threecysteine-rich subdomains, but not the fourth cysteine-rich subdomain, ofthe extracellular binding domain of a receptor selected from the groupconsisting of the 55 kD and 75 kD receptors for human TNFα.
 8. Acomposition according to claim 7, wherein said polypeptide has the aminoacid sequence of SEQ ID NO:
 2. 9. A composition according to claim 7,wherein said polypeptide has the amino acid sequence of SEQ ID NO: 4.